INTRODUCTION

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Immature T cell clones in the thymus are selected to survive or die at the CD4⁺CD8⁺ stage according to the specificity of the T cell receptors (TCRs).¹ Useful clones are protected from apoptosis and differentiate into mature CD4⁺CD8 or CD4CD8⁺ T cells (positive selection), while self-reactive clones undergo apoptosis (negative selection). Useless clones also appear to undergo apoptosis. The TCR-mediated signals are critical for the fate of CD4⁺CD8⁺ thymocytes and involve protein kinase C (PKC) activation. PKC consists of several subfamilies of enzymes including Ca²⁺-dependent classical PKC (cPKC) and Ca²⁺-independent novel PKC (nPKC) and atypical PKC (aPKC) (1). Each isoform or subfamily of PKC appears to play its own role in the fate of CD4⁺CD8⁺ thymocytes. We have previously indicated that activation of cPKC is involved in positive selection (2). On the other hand, thymocyte apoptosis induced by TCR/CD3- and CD28-mediated stimulation in vitro appears to be accompanied by activation of both cPKC and nPKC,² and the death is considered to mimic negative selection (3).

Glucocorticoid hormones exert pleiotropic effects on thymocyte survival and differentiation (4-10) and may participate in apoptosis of unselected thymocyte clones. CD4⁺CD8⁺ thymocytes are highly sensitive to induction of apoptosis by glucocorticoids and appear to undergo apoptosis even at the physiological peak levels at least in mice or rats (5, 6, 10, 11), whereas immature CD4CD8 thymocytes or mature CD4⁺CD8 or CD4CD8⁺ thymocytes are relatively resistant (12, 13). Glucocorticoid-induced apoptosis in thymocytes is preceded by activation of nPKC including nPKC- and is inhibited by non-isoform-selective PKC inhibitors but not by Gö 6976, a specific inhibitor of cPKC isoforms and PKC-µ (11, 16, 17). The apoptosis is also inhibited by proper levels of stimulation through TCR·CD3 complex with co-stimulation through CD4, CD8, or lymphocyte function-associated antigen-1 (11, 16, 17). The antiapoptotic effect is mimicked by moderate stimulation with proper combinations of PMA and the Ca²⁺ ionophore ionomycin or combinations of thymeleatoxin (TTX) and ionomycin (2, 18, 19). PMA activates both cPKC and nPKC (20), while TTX at the antiapoptotic doses specifically activates cPKC (2, 20, 21). On the other hand, the cPKC (and PKC-µ)-specific inhibitor Gö 6976 (22, 23) cancels the antiapoptotic effect of the antibodies and that of PMA/ionomycin (13). Gö 6976 also inhibits positive selection in a fetal thymus organ culture system (2). Therefore, proper levels of cPKC activity appear to be involved in both the protection of CD4⁺CD8⁺ thymocytes from apoptosis and the induction of positive selection. Indeed, transient stimulation of isolated CD4⁺CD8⁺ thymocytes with the antiapoptotic combinations of TTX/ionomycin induced differentiation and commitment of the cells to the CD4 or CD8 T cell lineage (2).

In the present study, we analyzed a possible relationship between thymocyte apoptosis and activation of nPKC isoforms by using the diterpene esters ingenol 3,20-dibenzoate (IDB) and TTX. We also analyzed the effect of calcineurin activation on nPKC-dependent apoptosis in thymocytes, since activation of calcineurin as well as cPKC was critical for the inhibition of glucocorticoid-induced apoptosis in thymocytes and for thymocyte positive selection (16, 18). The present results suggest that nPKC activation induces apoptosis in immature CD4⁺CD8⁺ thymocytes and that calcineurin activation contributes to protect the cells from the apoptosis.

	EXPERIMENTAL PROCEDURES

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Mice and Reagents-- BALB/c mice (4-6 weeks of age) were obtained from Japan SLC (Shizuoka, Japan). BOG8 TCR transgenic mice with RAG-2(/), and nonselecting MHC backgrounds were established as described previously (19, 24). Major histocompatibility complex class I and class II double knockout (DKO) mice (C57BL/6 deficient in A^b and ₂-microglobulin) were obtained from Taconic (Immuno-Biological Laboratories, Gunma, Japan). DEX, PMA, and IDB were obtained from Wako (Osaka, Japan), Sigma, and Biomol (Plymouth Meeting, PA), respectively. Ionomycin, TTX, Gö 6976, Gö 6983, and recombinant human cPKC- and nPKC- were obtained from Calbiochem. H-7 and HA1004 were obtained from Seikagaku Kogyo (Tokyo, Japan). Ro 31-8425 (bisindolylmaleimide X) and Ro 31-6045 (bisindolylmaleimide V) were obtained from LC Laboratories (Läufelfingen, Switzerland).

Cell Culture-- Splenic T cells were obtained from BALB/c mice as described previously (16). In the splenic T cell preparations, 88-92% of the cells were TCR⁺ by fluorescence-activated cell sorting analysis. Thymocytes or splenic T cells (3.75-4 × 10⁶) were suspended in 1 ml of Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum (JRH Bioscience, Woodland, CA), 3 mM L-glutamine, 1 mM sodium pyruvate, 1× minimal essential medium nonessential amino acids, 50 µM 2-mercaptoethanol, 20 mM HEPES (pH 7.2), 20 units of penicillin, and 20 µg of streptomycin (complete Dulbecco's modified Eagle's medium) and were cultured for the indicated times at 37 °C in the presence or absence of the indicated drugs in 24-well tissue culture plates (Corning 25820, Corning, NY).

Fluorescence-activated Cell Sorting Analysis-- To examine the expression of CD4, CD8, and TCR, the cells were stained with labeled antibodies: R-phycoerythrin-conjugated anti-CD4 monoclonal antibody (RM4-5), fluorescein isothiocyanate-labeled anti-CD8 monoclonal antibody (53-6.7), or fluorescein isothiocyanate-labeled or biotinylated anti-TCR monoclonal antibody (H57-597) (Pharmingen) with or without streptavidin TRI-Color (Caltag Laboratories, San Francisco, CA). Viable cells were gated by using forward and side scatters with a FACScan flow cytometer and FACScan research software (Becton Dickinson, Lincoln Park, NJ) and were analyzed for marker expression. The gate for viable cells was determined by using propidium iodide exclusion and Paint-a-Gate software (Becton Dickinson).

Assays for DNA Fragmentation and Cytolysis-- DNA fragmentation in thymocytes was determined as described previously (11). Briefly, the cells harvested by centrifugation were lysed in 0.5% Triton X-100 containing 5 mM Tris-HCl (pH 7.4) and 1 mM EDTA for 20 min on ice. The lysate and its supernatant after centrifugation at 27,000 × g for 20 min were sonicated for 15 s, and then DNA contents were measured by fluorometry using 4',6-diamidino-2-phenylindole (Sigma) and Fluoroskan II (Titertek; Flow Laboratories USA, McLean, VA). The percentage of DNA fragmented was calculated as the ratio of DNA content in the supernatant to that in the lysate. Cytoysis was assessed by a trypan blue dye exclusion assay.

Western Blotting-- Immunoblotting analysis of PKC isoforms was performed as described previously (14) with slight modification. The cultured thymocytes were centrifuged at 450 × g for 5 min at 4 °C, and each cell pellet (10⁷ cells) was washed with ice-cold phosphate-buffered saline and resuspended in 1 ml of ice-cold buffer A (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.3% mercaptoethanol, 50 µg/ml phenylmethylsulfonyl fluoride, 250 µg/ml leupeptin, and 10 mM benzamidine). After a 5-min incubation, cell lysis was confirmed by trypan blue, and the suspension was centrifuged at 100,000 × g for 70 min. After centrifugation, the supernatant (cytosolic fraction) was removed, and the pellet was resuspended in 1 ml of buffer A supplemented with 1% Triton X-100 and sonicated for 1 min. The homogenate was applied onto DEAE-cellulose DE-52 columns for removing DNA and partial purification. The columns were washed with ice-cold buffer B (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.3% mercaptoethanol, 50 µg/ml phenylmethylsulfonyl fluoride, and 10 mM benzamidine) and eluted with buffer B supplemented with 0.2 M NaCl. The eluate is referred to as the particulate fraction. The proteins in the particulate fractions and those in the cytosolic fractions were precipitated with ethanol (final concentration of 60% (v/v)). Equivalent amounts of proteins were solubilized in sample buffer with 2-mercaptoethanol and separated by SDS-polyacrylamide gel electrophoresis (9% gel) and transferred to nitrocellulose membranes (Micron Separations, Westboro, MA). The membranes were soaked in 5% bovine serum albumin and incubated with monoclonal anti-PKC antibodies (Transduction Laboratories, Lexington, KY). PKC isoforms (, , , , , , , µ, , and ) were detected with horseradish peroxidase-goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and the ECL system (Amersham Pharmacia Biotech, Tokyo, Japan). It should be noted, however, that the anti-cPKC- cross-reacts with  and that the anti-cPKC- cross-reacts with  according to the manufacturer.

Transmission Electron Microscopy-- Cells were prefixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), washed twice with 0.1 M phosphate buffer, postfixed with 2% osmium tetroxide in 0.1 M phosphate buffer, and embedded in 2% agar. The cells were dehydrated in an ethanol series, embedded in Epon 812, and kept at 60 °C for more than 48 h to polymerize the resin. After ultrathin sections were stained with uranyl acetate and lead citrate, they were observed under 1200EX transmission electron microscopy (JEOL, Tokyo, Japan).

	RESULTS

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Selective Activation of nPKC Isoforms in Thymocytes upon Stimulation with DEX-- We have previously shown that glucocorticoids induce an increase in nPKC activity in the particulate fraction of murine thymocytes and translocation of nPKC- from the cytosolic fraction to the particulate fraction (14). Other nPKC isoforms were not significantly detected in these cells by using the antibodies available at that time, but here we could also detect nPKC- and - by using newly available antibodies. nPKC- was not detectable. In the normal thymus, 80-85% of thymocytes are immature CD4⁺CD8⁺ cells that express low levels of Bcl-2 and are sensitive to induction of apoptosis by glucocorticoids (25). To obtain CD4⁺CD8⁺ thymocytes, DKO mice were used, since T cell development is arrested at the CD4⁺CD8⁺ stage in these mice, and almost all of the thymocytes are CD4⁺CD8⁺ cells (26). The synthetic glucocorticoid, DEX, induced increases in nPKC- and - in the particulate fraction of thymocytes from DKO mice after 2-2.5 h of incubation and induced decreases in these isoforms in the cytosolic fraction after 2-3 h of incubation (Fig. 1), suggesting that the nPKC isoforms were translocated. cPKC isoforms in the particulate fraction only slightly increased after 2 h of incubation, while those in the cytosolic fraction did not significantly change (Fig. 1). DEX did not induce translocation of PKC-µ, a PKC distantly related to nPKC (1), or that of aPKC- or - (Fig. 1 and data not shown). Similar changes in the intracellular distribution of PKC isoforms were observed in BALB/c mouse thymocytes treated with DEX, and the translocation of nPKC- was also confirmed (data not shown). However, the expression of nPKC- molecule in C57BL/6 and DKO thymocytes was consistently low, and thus its translocation was hardly detectable. Since DEX-induced DNA fragmentation in thymocytes is inhibited by non-isoform-selective PKC inhibitors but not by the cPKC-specific inhibitor Gö 6976 (14, 15), the results suggest that activation of nPKC isoforms may be involved in the death but that nPKC- activation may not be essential.

View larger version (78K): [in this window] [in a new window]   Fig. 1.   DEX induces selective translocation of nPKC isoforms in CD4+CD8+ thymocytes. Thymocytes from DKO mice were cultured with 100 nM DEX for 0, 2, 2.5, or 3 h and fractionated into the cytosolic fractions and the particulate fractions. Equivalent amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. PKC isoforms were detected by Western blot analysis with antibodies to cPKC-, -, and -; nPKC- and -; PKC-µ; or aPKC-. To compare relative amounts of proteins in each lane, proteins were stained with Coomassie Brilliant Blue R-250 (CBB), and the protein bands in an arbitrary range were shown. A representative result of four independent experiments is shown.

Selective Activation of nPKC Isoforms in Both Thymocytes and Splenic T Cells with IDB and Dose-dependent Activation of PKC Subfamilies with TTX-- The diterpene diester IDB has once been suggested to be a selective activator of PKC-, but it is not specific to this isoform as the manufacturer indicates in the product catalog. IDB at 10 or 50 nM induced translocation of nPKC-, -, and - and PKC-µ from the cytosolic fractions to the particulate fractions of both thymocytes and splenic T cells, whereas it did not induce translocation of cPKC isoforms or aPKC- and - (Fig. 2 and data not shown), indicating that IDB selectively activates nPKC isoforms and PKC-µ in these cells in this dose range. On the other hand, as TTX is known as a cPKC-specific activator (20, 21), incubation of thymocytes with 0.3 ng/ml TTX induced selective translocation of cPKC- and - (Fig. 2). However, at higher concentrations (1 ng/ml or higher), TTX is no longer specific for cPKC in thymocytes. Incubation of the cells with 1 ng/ml TTX induced translocation of nPKC- and -µ as well as cPKC isoforms (Fig. 2). There was little translocation of nPKC- or - upon stimulation with 1 ng/ml TTX. The translocational response of each PKC isoform in thymocytes is summarized in Table I.

View larger version (75K): [in this window] [in a new window]   Fig. 2.   IDB induces selective translocation of nPKC isoforms in thymocytes, while TTX dose dependently induces translocation of various PKC isoforms. Thymocytes from BALB/c mice were incubated with 0, 0.3, or 1 ng/ml TTX or with 10 or 50 nM IDB for 20 min at 37 °C. The cells were then fractionated and analyzed for the subcellular distribution of PKC isoforms as described in the legend to Fig. 1. To compare relative amounts of proteins in each lane, proteins were stained with Ponceau S, and the protein bands in an arbitrary range were shown. A representative result of four independent experiments with similar design is shown.

Induction of Apoptosis with IDB or High Concentrations of TTX in Thymocytes but Not in Splenic T Cells-- IDB induced DNA fragmentation in thymocytes but not in splenic T cells (Fig. 3, A and B), indicating that IDB induces death in immature T cells but not in mature T cells. IDB as well as DEX induced DNA fragmentation in CD4⁺CD8⁺ thymocytes from DKO mice and BOG8 TCR transgenic mice with RAG-2 (/) and nonselecting major histocompatibility complex backgrounds (data not shown). In the latter mice, T cell development is also arrested at CD4⁺CD8⁺ thymocytes, and almost all of the thymocytes are CD4⁺CD8⁺ cells (19) as in DKO mice. Incubation of thymocytes with IDB induced morphological changes typical in apoptosis, such as shrinkage of cells, clumping of chromatin into masses, and dilation of endoplasmic vesicles, while keeping mitochondrial structure almost normal (Fig. 4) as observed in glucocorticoid-treated thymocytes (4). TTX at 0.3 ng/ml did not induce DNA fragmentation in thymocytes, but TTX at 1 ng/ml did induce it (Fig. 3C). Thus, there was a close correlation between activation of nPKC, especially the -isoform, and induction of apoptosis in thymocytes.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   IDB induces DNA fragmentation in thymocytes but not in splenic T cells, while TTX at 1 but not 0.3 ng/ml induced DNA fragmentation in thymocytes. Thymocytes (A, B, and C) or splenic T cells (A) were cultured with graded concentrations of IDB (A and B) or TTX (C) for 16 h. After the culture, DNA fragmentation was assessed. Data are expressed as means ± S.D. of triplicate cultures.

View larger version (125K): [in this window] [in a new window]   Fig. 4.   Morphologically typical apoptosis is induced in thymocytes by IDB stimulation. Thymocytes from BALB/c mice were cultured in the presence (D, E, and F) or the absence (A, B, and C) of 50 nM IDB for 16 h. After the culture, the cells were harvested and fixed, and their morphology was analyzed by electron microscopy.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   A time lag and macromolecular synthesis are required for inducing DNA fragmentation in IDB-treated thymocytes. A, BALB/c mouse thymocytes were cultured with 50 nM IDB in the presence or absence of 1 µM actinomycin D (Act. D) or 10 µM cycloheximide (CHX) for 16 h. B, BALB/c mouse thymocytes were cultured with or without 50 nM IDB for 3, 6, or 16 h. The cultured cells were assessed for DNA fragmentation. Data are expressed as means ± S.D. of triplicate cultures.

Involvement of nPKC Activation in IDB-induced Thymocyte Apoptosis-- PKC inhibitors were used to examine if PKC activation is essential for the induction of apoptosis. H-7, an inhibitor of both nPKC and cPKC and some other kinases (27, 28), inhibited IDB-induced DNA fragmentation in thymocytes (Fig. 6A). Ro 31-8425, a staurosporine-related and highly specific PKC inhibitor (29), also inhibited the DNA fragmentation (Fig. 6A). The inhibition of death was confirmed by a trypan blue dye exclusion assay (data not shown). Ro 31-8425 at 1 µg/ml completely inhibited the activation of recombinant cPKC- and recombinant nPKC- in vitro (data not shown), confirming that Ro 31-8425 is a non-isoform-selective PKC inhibitor. On the other hand, the structural analogs of these inhibitors with weak PKC-inhibitory activity, HA1004 and Ro 31-6045 (27, 30), did not inhibit DNA fragmentation (Fig. 6A). The selective inhibitor of cPKC and PKC-µ, Gö 6976, also failed to inhibit the IDB-induced DNA fragmentation, whereas its structural analog, Gö 6983 inhibited the death (Fig. 6B). Gö 6983 has been shown to inhibit various PKC isoforms including cPKC, nPKC-, and PKC-, but it does not effectively inhibit PKC-µ (23). Similar effects were observed on TTX-induced DNA fragmentation in thymocytes by using these inhibitors and analogs (Fig. 6B and data not shown). The results collectively suggest that nPKC activation is essential for the induction of thymocyte apoptosis by IDB.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   IDB- or TTX-induced DNA fragmentation in thymocytes is inhibited by non-isoform-selective PKC inhibitors but not by the cPKC- and PKC-µ-specific inhibitor Gö 6976. A, thymocytes from BALB/c mice were cultured for 16 h in the presence of 50 nM IDB with or without 40 µM H-7, 40 µM HA1004, 1 µM Ro 31-8425, or 1 µM Ro 31-6045. B, BALB/c mouse thymocytes were cultured for 16 h in the presence of 50 nM IDB or 1 ng/ml TTX with or without 3 µM Gö 6976 or Gö 6983. After the culture, the cells were assessed for DNA fragmentation. Data are expressed as means ± S.D. of triplicate cultures.

Calcineurin-dependent Inhibition of IDB-induced Apoptosis in Thymocytes by a Proper Increase in the Intracellular Ca²⁺ Level-- Activation of calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, contributes to the inhibition of glucocorticoid-induced thymocyte apoptosis (16, 18) and is essential for positive selection (16, 31, 32). Thus, we examined if calcineurin activation also contributes to the inhibition of IDB- or TTX-induced apoptosis in thymocytes. Ionomycin alone induced DNA fragmentation in thymocytes in a dose-dependent fashion but inhibited IDB-induced DNA fragmentation at concentrations within a narrow range (0.2-0.3 µg/ml) (Fig. 7A). Thus, ionomycin at these concentrations and IDB were mutually antagonistic in the induction of apoptosis. Similar results were obtained with TTX-induced DNA fragmentation (data not shown). The inhibition of apoptosis was canceled by FK506 but not by rapamycin (Fig. 7B). Although the structurally related immunosuppressive macrolides FK506 and rapamycin commonly bind to FKBP-12, the FK506·FKBP-12 complex but not the rapamycin·FKBP-12 complex inhibits the phosphatase activity of calcineurin (33). The result thus suggests that calcineurin activation is essential for the antiapoptotic effect. Glucocorticoid-induced DNA fragmentation in thymocytes was not inhibited by ionomycin alone, but it was inhibited by 0.2-0.3 µg/ml ionomycin in the presence of 0.3 ng/ml TTX (Fig. 7C) (2). Activation of cPKC as well as calcineurin has been suggested to contribute to the inhibition of glucocorticoid-induced thymocyte apoptosis (2). Incubation of thymocytes with 0.2 µg/ml ionomycin alone induced little change in the distribution of PKC isoforms, but incubation with the combination of 0.2 µg/ml ionomycin and 50 nM IDB induced translocation of cPKC as well as nPKC (Fig. 8), suggesting that cPKC was indeed activated with ionomycin/IDB. It was also noted that IDB-induced translocation of nPKC was moderately reduced with ionomycin (Fig. 8). Thus, activation of calcineurin and cPKC may commonly contribute to the inhibition of glucocorticoid-induced apoptosis and IDB-induced apoptosis in thymocytes.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   IDB-induced DNA fragmentation is inhibited by ionomycin at the concentrations within a narrow range, and the inhibition is canceled by FK506 but not by rapamycin. A, BALB/c mouse thymocytes were cultured for 16 h with or without 50 nM IDB in the presence of graded concentrations of ionomycin (IM). B, BALB/c mouse thymocytes were cultured for 16 h with medium, 0.2 µg/ml ionomycin, 50 nM IDB, or 0.2 µg/ml ionomycin and 50 nM IDB in the presence or absence of 5 nM FK506 or rapamycin. After the culture, DNA fragmentation was assessed. Data are expressed as means ± S.D. of triplicate cultures.

View larger version (64K): [in this window] [in a new window]   Fig. 8.   The combination of ionomycin and IDB induces translocation of cPKC and moderately reduces translocation of nPKC. BALB/c mouse thymocytes were incubated with 0.2 µg/ml ionomycin and 50 nM IDB for 20 min at 37 °C. The cells were analyzed for the subcellular distribution of PKC isoforms as described in the legend of Fig. 1. To compare relative amounts of proteins in each lane, proteins were stained with Coomassie Brilliant Blue R-250 (CBB), and the protein bands in an arbitrary range were shown. A representative result of three independent experiments with similar design is shown.

	DISCUSSION

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Our previous study suggested that activation of nPKC is involved in glucocorticoid-induced apoptosis in BALB/c mouse thymocytes (14). Although the -isoform was detected as the major nPKC isoform in BALB/c mouse thymocytes in that study and in other studies (34, 35), we found here that the -isoform is only weakly detectable in thymocytes from normal or class I/class II-deficient C57BL/6 mice despite the fact that it is clearly detectable in the brain of these mice (data not shown). Here we detected the nPKC isoforms  and  and found that they were also activated with DEX in CD4⁺CD8⁺ thymocytes (Fig. 1). Glucocorticoid-induced thymocyte apoptosis is inhibited by non-isoform-selective PKC inhibitors but not by cPKC-specific inhibitors (14, 15). Thus, nPKC isoforms, not necessarily the -isoform, appear to be involved in the apoptosis. To confirm the possible involvement of nPKC activity in the induction of apoptosis, we cultured thymocytes with IDB and TTX. IDB induced selective translocation of nPKC-, -, and - and PKC-µ (Fig. 2) and induced apoptosis in thymocytes (Figs. 3 and 4). TTX at a relatively low concentration induced selective translocation of cPKC (Fig. 2) but failed to induce apoptosis (Fig. 3). However, TTX at relatively high concentrations induced translocation of cPKC, nPKC-, and PKC-µ and induced apoptosis (Figs. 2 and 3). The IDB- and TTX-induced apoptosis was inhibited by the non-isoform-selective PKC inhibitors but not by the selective inhibitor of cPKC and PKC-µ, Gö 6976 (Fig. 6). The dose of Gö 6976 was enough to cancel the antiapoptotic effect of the antibodies and the effect of the ionomycin/PMA on glucocorticoid-treated thymocytes (15). Thus, nPKC activation is likely to be responsible for the induction of thymocyte apoptosis. It remains unclear if nPKC isoforms have redundant effects on the induction of apoptosis or if one of the nPKC isoforms, especially , is solely responsible for the death.

Phorbol esters bind to the C1 domain of PKC, and the C1 domain is also found in some other proteins including Vav, a guanine nucleotide exchange protein required for positive selection (36). Vav is directly activated by PMA, but the activation is not inhibited by staurosporine (37). Staurosporine is a non-isoform-selective PKC inhibitor and does not bind to the C1 domain. Since thymocyte apoptosis induced by DEX, IDB, or TTX was inhibited by staurosporine or the staurosporine-related non-isoform-selective PKC inhibitors (Fig. 6) (14), it is unlikely that the death depends on Vav activation. It is still possible, however, that thymocyte apoptosis is induced through activation of an unknown molecule whose activity is regulated by the inhibitors and activators of PKC exactly as the nPKC activity is regulated.

A role of nPKC in apoptosis was suggested also in some human leukemic cell lines, in which active fragments of nPKC isoforms were induced by proteolysis upon stimulation with the apoptosis-inducing agents (38-40). On the other hand, activation of PKC with high doses of phorbol esters may trigger its ubiquitination and degradation by proteasome and may result in the depletion of PKC (39). In DEX-treated thymocytes, the amounts of nPKC- and - in the cytosolic fraction continuously decreased in a time-dependent fashion, while those in the particulate fraction increased within 2.5 h of incubation but decreased thereafter (Fig. 1), indicating that these PKC isoforms were degraded after activation. It has been shown that calpain inhibitors inhibit DEX-induced apoptosis in thymocytes (41). However, the active fragments of nPKC were not detected in thymocytes after 2-3 h of incubation with DEX (data not shown). The nPKC activities in the cytosolic and particulate fractions of DEX-treated thymocytes were dependent on both PMA and phospholipid (14), whereas the catalytic fragment of PKC produced by limited proteolysis with calpain is enzymatically active independent of the two (42).

Apoptosis in thymocytes at the CD4⁺CD8⁺ stage is critical for clonal selection of T cells. Even physiological peak levels of glucocorticoid hormones may induce or enhance death in CD4⁺CD8⁺ thymocytes (8-11), especially in unselected or useless clones. However, useful clones are likely to be protected from physiological levels of glucocorticoids by positive selection signals (11, 15). Accordingly, positively selected CD4⁺CD8⁺ thymocytes become relatively resistant to glucocorticoid-induced death (43). Proper stimulation through TCR/CD3 suppresses glucocorticoid-induced apoptosis in murine thymocytes (11), and costimulation through accessory molecules including CD4, CD8, or lymphocyte function-associated antigen-1 enhances the antiapoptotic effect partly because it enhances the TCR/CD3-mediated increase in the intracellular Ca²⁺ concentration (16, 44, 45). The antiapoptotic effect of the antibodies was mimicked by proper combinations of ionomycin and PMA or TTX, and transient stimulation of the cells with the combinations induced the early processes of positive selection: differentiation and commitment of the cells to the CD4 or CD8-T cell lineage (2, 19, 24). The antiapoptotic and differentiation-inducing effects appear to be dependent on activation of calcineurin and cPKC (2, 16, 18). The ionomycin concentrations have to be within a narrow range for these effects (Fig. 7C) (19). IDB-induced thymocyte apoptosis was inhibited by the same concentration range of ionomycin in the presence or absence of a cPKC activator (Fig. 7A and data not shown). The ionomycin/IDB treatment of thymocytes induced cPKC activation (Fig. 8). The inhibition of IDB-induced apoptosis as well as that of glucocorticoid-induced apoptosis was canceled by FK506 but not by rapamycin (Fig. 7B) (16), suggesting that calcineurin activation is essential for the antiapoptotic effect. Calcineurin regulates the activities of several transcription factors including the nuclear factor of activated T cells (NFAT), especially NFATx in CD4⁺CD8⁺ thymocytes (46). However, it is not known how the nPKC-dependent pathway and the calcineurin-dependent pathway interact each other. In some other cell types, calcineurin activation has been rather suggested to induce or enhance apoptosis (47). Excessive doses of ionomycin failed to inhibit IDB-induced thymocyte apoptosis. It may involve excessive activation of calcineurin or activation of other Ca²⁺-dependent enzymes.

Macromolecular synthesis is required for glucocorticoid-induced apoptosis and nPKC activation in thymocytes (14). Once mitochondrial permeability transition is induced, macromolecular synthesis is not required for apoptosis to occur (48). The expression of Nur77/Nurr1 and p53 are required for activation-induced apoptosis and radiation- or etoposide-induced apoptosis in thymocytes, respectively, but are not required for glucocorticoid-induced apoptosis (49-51). A death-inducing gene for glucocorticoid-induced thymocyte apoptosis has been postulated but not clarified. Since the IDB-induced apoptosis also requires macromolecular synthesis (Fig. 5), glucocorticoid-induced thymocyte apoptosis may depend on at least two steps of macromolecular synthesis before and after the nPKC activation. IDB also activated nPKC in mature T cells but did not induce apoptosis (Fig. 3A). Mature T cells express Bcl-2 and may possess an antiapoptotic mechanism against the "death-inducing gene" or may not be able to express the gene. A further insight into the mechanism of glucocorticoid-induced thymocyte apoptosis would be obtained by comparing glucocorticoid- and IDB-induced molecular events in thymocytes and mature T cells.